Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes

Lipids are key constituents of all cells, which express thousands of different lipid species. In most cases, it is not known why cells synthesize such diverse lipidomes, nor what regulates their metabolism. Although it is known that dividing cells specifically regulate their lipid content and that the correct lipid complement is required for successful division, it is unclear how lipids connect with the cell division machinery. Here, we report that the membrane protein stomatin is involved in the cytokinesis step of cell division. Although it is not a lipid biosynthetic enzyme, depletion of stomatin causes cells to change their lipidomes. These changes include specific lipid species, like ether lipids, and lipid families like phosphatidylcholines. Addition of exogenous phosphatidylcholines rescues stomatin-induced defects. These data suggest that stomatin interfaces with lipid metabolism. Stomatin has multiple contacts with the plasma membrane and we identify which sites are required for its role in cell division, as well as associated lipid shifts. We also show that stomatin’s mobility on the plasma membrane changes during division, further supporting the requirement for a highly regulated physical interaction between membrane lipids and this newly identified cell division protein.


CRISPR/Cas9 cell line
The creation of the stable cell line was performed as described by McKinley 1 with the following optimizations.
The single guide RNA (sgRNA) sequences were designed using http://crispr.mit.edu. The sgRNAs were cloned into pLentiGuide-Puro, a gift from Feng Zhang (Addgene plasmid # 52963), a two vector system characterized by a U6 promoter located before the sgRNA insertion site. To insert the single guide RNA in the plasmid, a double   Figures S1F and S6B), or 100x oil objective (for images in Figure S6A).
For live cell imaging, cells lines expressing fluorescent proteins were plated on imaging μ-slide or 24 well μplates (Ibidi). Cells were transfected with siRNAs and imaged 30-34h after transfection in FluoroBrite media supplemented with 10% FBS, L-glutamine and sodium pyruvate (Life Technologies). Movies were acquired on Nikon Eclipse microscopes attached to widefield epifluorescence (Ti-E) (for Movie S1 and for time-lapse in Figure S1) or Confocal A1R imaging systems with environmental chambers to maintain cells at 37°C and 5% CO2 (for FRAP experiments in Figure S7 and Movie S2). Frames were acquired typically every 3 min unless stated otherwise. Images were processed using NIS elements software (Nikon) and adjusted for brightness using Image J or Photoshop CS5.1 (Adobe). concentration and samples were then boiled (100°C) for 10 min. Proteins were resolved using SDS-PAGE followed by wet-tank transfer onto nitrocellulose membrane (0.22 μm, Biorad). Membranes were probed with the indicated antisera in 5% milk in PBS-T overnight at 4°C, prior to washing and re-incubation with secondary HRPconjugated antibodies (Jackson ImmunoResearch).

Fluorescence recovery after photobleaching (FRAP)
HeLa cells stably expressing the different Stomatin constructs were plated in a 24 well ibidi imaging plate (μ-Plate ibidi) and incubated for 24 h. FRAP analysis was conducted using a Nikon inverted Confocal A1R imaging system using 488 nm laser for FITC excitation, in addition the microscope is equipped with a stage heater at 37°C.
The acquisition was performed using a 60x oil lens objective with a frame size of 512x512 pixels. A zoom of 2.39 pixels (size: 0.19 µm) was applied. For the FRAP acquisition, a Region of Interest (ROI) of 3x3 µm was selected.
The pinhole was set at 1.2 AU. For acquisition, the laser power was equally set for every specimen tested, since a variable laser power setting for could introduce unwanted bias. Some transgenic proteins were more stable than others, and hence the gain control was set appropriately. The laser power was set at 7.0, and the gain was adjusted from about 600 to 850 depending on the cell line tested. 91 images (each scan represented 3 sec elapsed time) were taken for each cell. The bleach starts after 3 scans of 1 second each. 3 The images were analyzed using FIJI software and the following pipeline was optimized following Wachsmuth's work. 4 For each image 3 different round areas (ROI) were drawn of the same size (3x3µm) of the bleach region that was used during the acquisition.
The area selected was including: This eliminates noisy signal from the data.
2. Normalize corrected Bleach to corrected Reference.
3. Normalize to the mean pre-bleach intensity.
At this point, the data were plotted using the Curve Fitting tool of FIJI. The fitting was based on the "Exponential Recovery" features: UHPLC analytical gradient was set to: B from 15% to 30% (0-2 min): B from 30% to 48 % (2-2.5 min); B from 48% to 82% (2.5-11 min); B from 82% to 99% (11-11.5 min) and B kept on 99% for 4 minutes in order to wash out the non-polar compounds. In 0.5 min B returned to its initial conditions and the column was equilibrated for 3 minutes for the next run. Sample needle was washed with 100% isopropanol between injections. Samples were ordered randomly, blank extractions and pooled (QC) extractions were also added to sample sequence for further quality control steps.
Q-TOF Acquisition Parameters: The instrument was calibrated for both polarities and analyses were performed in the extended dynamic range mode (∼2 GHz) in the mass range 50 to 1700 m/z. Hexakis phosphazene and purine molecules were used as calibrants during the analyses.
Instrument Parameters: The

Targeted data analysis by in-house lipid library
We employed our in-house lipid library as a complimentary peak selecting approach in order to investigate the lipid changes in more detail (Table S2) (Table S2 and Fig. 2), which did not necessarily pass the Fold Change reduction process. For the lipidomic data obtained from the cell lines expressing different GFP-Stomatin constructs we monitored 500 lipid species over 15 lipid classes (Fig. 4A). All the curated lipids were statistically tested with the same parameters as the untargeted analysis. Significance of targeted lipids are also shown in Table S2, if the p score is calculated higher than 0.05, the lipid is labelled as N.S. (non-significant).
Previously identified lipids from the untargeted analysis were added to these files if they were not previously included in the library. In targeted analysis, peak integration was confirmed manually, which caused insignificant differences between the area of the peaks in targeted and untargeted analyses, as seen in Tables S1 vs. S2.

Identification of PIPs
The PIPs were extracted using a modified version of the Folch method (CHCl3/MeOH 2:1, v/v) as described by Lysates were centrifuged at slow speed (3,500 xg for 5 min). Anti-GFP magnetic agarose beads (Chromotek) were washed, before equilibration, twice with ice-cold PIPES buffer (25 μL of beads were used for each sample).
Lysates were equilibrated for 1 hour at 4°C with anti-GFP beads in tube rotators. After rotation, supernatants were discarded, and beads were washed twice with 500 µL ice-cold PIPES buffer. Lipids were extracted by adding 250

ViaLight™ plus assay
HeLa cells were plated in 96-well white walled plates (Corning™ Costar™) and proceeded to ATP level detection 72 h after siRNA transfection (see methods above). Then, cells were cooled to room temperature and washed three times using PBS. ViaLight™ plus kit (Lonza) was used to detect the ATP levels in cells according to manufacturer's instruction. Briefly, 50 µL of cell lysis reagent was added to each well and incubated for 10 min at room temperature. After the incubation, 100 µL of pre-prepared ATP Monitoring Reagent Plus (AMR Plus) was added to cells and incubated for 2 min in the dark. The ATP level in cells was then read and recorded by plate reader ClarioStar using its luminescence detection mode.

PC rescue experiment
Cells were supplemented with bovine Liver PC (840055, Avanti Polar Lipids). The lipid mix was resuspended in methanol/water 95:5 (0.5 mg/mL), then heated and sonicated. The solution was then transferred to a glass vial and dried using a stream of nitrogen. The pellet was resuspended in a BSA solution (4 mg/mL) to make a 125 µM lipid solution and incubated for 30 minutes at 37 °C to facilitate complete solution. The PC mix was then added to cells after 24h of transfection with siRNA (siNT and siSTOM 1, see method above) and left in the media for 48h.

Statistical analysis and data presentation
For counts of multinucleated cells mean and standard deviation of the mean was calculated for N >=3 independent experiments unless otherwise stated (typically >300 cells were counted for each data point experiment unless otherwise stated). Bar graphs were drawn using GraphPad Prism. For timing throughout division experiments, Group Column Scatter plots of the data are presented showing median value, graphs were drawn using GraphPad Prism. Statistical significance was assessed using an unpaired two-tailed Student's t-test assuming equal variance.       The table shows the basic features including    Movie S3. GFP-Stomatin WT and its mutants localize at the plasma membrane during cell division.

Supplementary Table and Movie Legends
HeLa cells stably expressing GFP-Stomatin WT, C30S or dC (respectively left, center and right) imaged after 24 h seeding. Frames were acquired every 5 minutes, playback speed is 10 frames/second, scale bar = 10µm.